The effects of chronic acetaminophen exposure on the kidney, gill and liver in rainbow trout (Oncorhynchus mykiss)

Accepted Manuscript Title: The effects of chronic acetaminophen exposure on the kidney, gill and liver in rainbow trout (Oncorhynchus mykiss) Authors: Eugene Choi, Derek Alsop, Joanna Y. Wilson PII: DOI: Reference:

S0166-445X(18)30117-6 https://doi.org/10.1016/j.aquatox.2018.02.007 AQTOX 4855

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

24-11-2017 7-2-2018 8-2-2018

Please cite this article as: Choi, Eugene, Alsop, Derek, Wilson, Joanna Y., The effects of chronic acetaminophen exposure on the kidney, gill and liver in rainbow trout (Oncorhynchus mykiss).Aquatic Toxicology https://doi.org/10.1016/j.aquatox.2018.02.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The effects of chronic acetaminophen exposure on the kidney, gill and liver in

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rainbow trout (Oncorhynchus mykiss)

Eugene Choi, Derek Alsop and Joanna Y. Wilson

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Department of Biology, McMaster University, Hamilton ON Canada.

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*Address correspondence to: Joanna Y. Wilson

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Department of Biology, McMaster University

1280 Main St. West, Hamilton, ON, Canada, L8S 4K1

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Fax: (905) 522-6066

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Tel: (905) 525-9140 extension 20075

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EMAIL: [email protected]

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Research Highlights

Rainbow trout were exposed to 0, 10 and 30 µgL-1 acetaminophen for 4 weeks

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Acetaminophen impacted kidney, gill and liver histology

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Acetaminophen impacted kidney, gill and liver function

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Acetaminophen altered urine ion, glucose and protein concentrations

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Exposure limited oxygen consumption and decreased critical swimming speed

ABSTRACT In this study, we examined if rainbow trout chronically exposed to acetaminophen (10 and 30 μgL-1) showed histological changes that coincided with functional changes in the kidney,

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gill and liver. Histological changes in the kidney included movement and loss of nuclei, nonuniform nuclei size, non-uniform cytoplasmic staining, and loss of tubule integrity. Histological effects were more severe at the higher concentration and coincided with concentration dependent increases in urine flow rate and increased urinary concentrations of sodium, chloride, potassium, calcium, urea, ammonia, glucose, and protein. Yet, glomerular filtration rate was not altered with acetaminophen exposure. In the gill, filament end swelling, whole filament swelling, and

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swelling of the lamellae were observed in exposed fish. Lamellar spacing decreased in both

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exposure groups, but lamellar area decreased only with 30 µgL-1 exposure. At faster swimming speeds, oxygen consumption was limited in acetaminophen exposed fish, and critical swimming

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speed was also decreased in both exposure groups. The liver showed decreased perisinusoidal

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spaces at 10 and 30 µgL-1 acetaminophen, and decreased cytoplasmic vacuolation with 30 µgL-1 acetaminophen. A decrease in liver glycogen was also observed at 30 µgL-1. There was no

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change in plasma concentrations of sodium, chloride, potassium, calcium, magnesium, and glucose with exposure, suggesting compensation for urinary loss. Indeed, an increase in Na+-K+-

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ATPase activity in the gills was found with 30 µgL-1 acetaminophen exposure. Chronic exposure of rainbow trout to the environmentally relevant pharmaceutical acetaminophen, alters both

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histology and function of organs responsible for ion and nutrient homeostasis.

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Keywords: pharmaceutical, paracetamol, analgesic, histology, homeostasis

INTRODUCTION

Many studies have documented pharmaceutical and personal care products in wastewater effluent and standing waters with concentrations typically in the range of ngL-1 to μgL-1 (Kolpin et al., 2002; Metcalfe et al., 2003, 2010; Galus et al., 2013a; Zuccato et al., 2000). The main pathway of pharmaceuticals to the environment is from human excretion after ingestion (Fent et al., 2006). Pharmaceuticals and personal care products encompass a broad range of compounds,

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spanning numerous chemical classes, and thus have large variability in function, structure, behaviour, and activity. Our study focused on the analgesic acetaminophen due to its frequent use and wide-spread detection in aquatic systems. For example, acetaminophen has been detected in surface waters in the U.S. with a median concentration of 0.11 µgL-1, a maximum concentration of 10 µgL-1 and a frequency of detection of 23.8% (Kolpin et al., 2002). In Canadian wastewater treatment plant effluents, the highest reported concentration of this

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compound was 62 µgL-1, with a frequency of detection of 58% (Guerra et al., 2014).

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Studies have shown histological changes after chronic, low dose pharmaceutical exposure

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in multiple organ systems of fish. For instance, rainbow trout exposed to the analgesic diclofenac (1 to 500 μgL-1) showed severe alterations in the gills, kidney and liver (Schwaiger et al., 2004).

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At the lowest observed effect concentration (LOEC) of 5 µgL-1, the pillar cells of the gill had undergone necrosis, while renal lesions, degeneration of tubular epithelia, and interstitial

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nephritis were found in the proximal and distal kidney tubules (Schwaiger et al., 2004). In

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another study, rainbow trout exposed to 5 µgL-1 diclofenac showed collapse of cellular compartmentalization, organelle disarrangement, and glycogen depletion in the liver (Triebskorn

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et al., 2004). Zebrafish exposed to acetaminophen, venlafaxine, carbamazepine, and gemfibrozil at 0.5 and 10 μgL-1 resulted in significant changes in proximal kidney tubule morphology (Galus

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et al., 2013b). These changes included loss of cytoplasmic staining, vacuolization, distortion of nuclei positioning, distortion of nuclei shape, loss of nuclei, loss of native structural integrity, and proteinaceous fluid surrounding the proximal kidney tubules (Galus et al., 2013b). However,

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no changes in the gill and the distal kidney tubules were observed in this species (Galus et al., 2013b). Other studies have documented changes in aspects of organ function following chronic, low dose pharmaceutical exposure in fish. Exposure of the anticonvulsant carbamazepine to rainbow trout caused a significant lowering of Na+-K+-ATPase (NKA) activity in the gills after

42 days (Li et al., 2009). Diclofenac exposure in rainbow trout caused a reducted expression of cyclooxygenase genes, COX-1 and COX-2, in the liver (Mehinto et al., 2010). Yet, studies that include an assessment of both histology and organ function after pharmaceutical exposure are lacking. The health implications of the histological changes have yet to be determined, and

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whether fish can compensate for pharmaceutical induced histological changes is not clear. In mammals, the analgesic properties of acetaminophen are due to inhibition of cyclooxygenase activity. In addition, high, acute doses are known to cause liver and kidney toxicity in mammals. In rodents, the acute liver failure has been attributed to mitochondrial damage and nuclear DNA fragmentation (McGill et al., 2012). More recent studies show that renal insufficiency occurs in approximately 1-2% of patients with acetaminophen overdose

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(Mazer and Perrone, 2008). Patients with acetaminophen toxicity and renal insufficiency had histological changes in the kidney, specifically, tubular epithelial cell necrosis in both the

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proximal and distal tubules (Bjorck et al., 1988). These high dose, acute effects in mammals

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appear to have overlap in organ and cell type to effects reported for low dose, chronic exposures

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to analgesics, including acetaminophen, in fish (Galus et al., 2013b; Schwaiger et al., 2004; Triebskorn et al., 2004).

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Using rainbow trout, we have focused our study on the histology and function of the kidney, gill, and liver after acetaminophen exposure. Fish gills are responsible for oxygen uptake

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and histological changes in this organ may impact this function. The kidney and gills are both involved in ion homeostasis through uptake and urinary reabsorption, respectively in fresh water

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fish (Zayed and Mohamed, 2004; Evans et al., 2005). The kidney is also responsible for nutrient homeostasis through the reabsorption of fuels such as glucose. The liver is heavily involved in

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macronutrient and xenobiotic metabolism, including glucose and glycogen metabolism and homeostasis (Xu et al., 2005; Mommsen, 1986; Nabb et al., 2006). We exposed rainbow trout to

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10 μgL-1 or 30 μgL-1 acetaminophen for four weeks to test whether any observed histological changes coincided with altered organ function.

MATERIALS AND METHODS Fish Care

Rainbow trout (350 g; Humber Springs Hatchery, Orangeville, ON, Canada), were housed in an opaque tank with a flow-through system of dechlorinated Hamilton tap water (moderately hard: [Na+] = 0.6 mequiv L-1, [Cl-] = 1.8 mequiv

L-1, [Ca2+] = 0.8 mequiv L-1,

[Mg2+] = 0.3 mequiv L-1, [K+] = 0.05 mequiv L-1; titration alkalinity 2.1 mequiv L-1, pH ~ 8.0; hardness ~ 140 mg L-1 as CaCO3 equivalents; temperature 12.5-15ºC, water flow rate = 5 L min). Fish were fed two times per week using commercial fish pellets (Martin Trout Aquaculture,

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Tavistock, Ontario, Canada; crude protein 45%, crude fat 9%, crude fibre 3.5%). Supplemental air and water flow rates were checked daily. Source water was chlorine tested weekly. Acetaminophen Exposure Experiments

For the time course experiment, rainbow trout (N=3 per tank and two tanks per treatment)

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were housed in 60 L opaque tanks and exposed to 0 µgL-1 (control) or 10 µgL-1 (66.2 nM)

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acetaminophen (Sigma-Aldrich, Oakville, Canada) for six weeks. Tank temperatures were maintained at 16°C. Exposure tanks were static and a 90% water change-out followed by re-

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dosing of acetaminophen was performed every 72 h. Water quality was tested weekly for pH,

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nitrate, nitrite, and ammonia; temperature was monitored daily. One fish from each tank was sampled every 2 weeks and liver, gill, and kidney tissues were dissected for histology (described

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below). Whole experiments were replicated three times to provide a N=6 fish at each time point

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and treatment.

Following the time course exposure, rainbow trout were housed in 60 L tanks and

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exposed to 0 µgL-1 (control), 10 µgL-1 (low; 66.2 nM), or 30 µgL-1 (high; 198.5 nM) for 4 weeks, as described above. This experiment was repeated 5 times for a maximum of N=15 for some treatments. After 4 weeks, fish were challenged in swim performance tests and/or cannulated to

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determine kidney function (described below). After physiological testing, fish were euthanized and blood was sampled using heparinized syringes and plasma was prepared by centrifugation at

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21,155xg for 10 minutes and then stored at -20°C. Gill, liver and kidney samples were either formalin-fixed or snap frozen in liquid nitrogen and stored at -80°C. Acetaminophen concentrations in the water were determined over 72 h in a separate experiment. Tanks (N=4) were set-up in the same manner as the exposures, except there were no fish. Tanks were dosed with acetaminophen, left for 3 days, then 90% of the water was changed, similar to the exposure. At this point, water samples (12 mL) were taken at 15 min, 24 h, 48 h

and 72 h after dosing. Control samples (0 µgL-1) were taken at 15 min. Samples were stored at 80°C until analysis. Histology Tissues were fixed in 10% neutral buffered formalin for 72 h and transferred to 70%

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ethanol. The samples were embedded in paraffin and sections were taken at 5 µm and mounted on superfrost glass slides (Fisher Scientific, Ottawa, ON). Sections were stained with hematoxylin and eosin Y (Richard-Allan Scientific, Kalamazoo, USA), following standard histology procedures and examined using a Zeiss Axiolab microscope (Carl Zeiss, Hallbergmoos, Germany). A modified scoring system adapted from Bernet et al. (1999) was used to identify the histological changes in the kidney. Briefly, kidney histological changes were scored from 0-4, 0

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representing no damage to the kidney, 1 representing nuclei movement, 2 for increased nuclei

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movement and multi-nucleation and loss of nuclei, 3 for heavy globular eosin staining and tubule damage, and 4 for heavy tubule degradation. A scoring system was developed for the gills based

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on histological changes observed by Mallat (1985). Gill histological changes were scored on a

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scoring system from 0-3 based on number of histological changes (filament tip swelling, thickening of lamellae, and whole filament swelling) observed, 0 indicating no change, 1

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representing at least one histological change, 2 representing at least two histological changes, and 3 indicating all three possible alterations were observed. Liver histological changes were not

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scored numerically, but were visually assessed for histological changes such as a decrease in perisinusoidal space, vacuolation of the cytoplasm, and glycogen depletion. The tissue histology

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slides were prepared and assigned a random numerical code by a third party so that the assessors were unaware of the treatment from which the sample originated until all assessments were

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completed (Galus et al., 2013b). The slides were read by a single assessor (E. Choi) and the histological changes were noted and confirmed by a second assessor (J. Wilson).

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Swimming Performance Swim tests were performed using a Loligo 90 L swim tunnel respirometer (Loligo

Systems, Tjele, Denmark) in order to determine oxygen consumption at increasing swimming speeds. Fish were allowed to acclimate for 30 minutes at 0.3 body-lengths/second (BLs-1) in the dark, while the tunnel was flushed continuously with fresh water. Swim tests began at 1.5 BL s-1 and increased by 0.5 BL every 20 min. During the test period, the swim chamber was sealed and

oxygen concentrations were monitored. Oxygen concentrations did not fall below 70% during these periods. The respirometer was flushed with fresh water prior to increasing the swimming speed for the next step. Oxygen content of the swim tunnel was measured using a fibre-optic oxygen probe and Loligo Autoresp LDAQ software (Loligo Systems). Metabolic rate was calculated using the linear regression of oxygen concentration of the water in the swim tunnel,

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along with the fish mass, flume volume corrected for fish volume, and saturation concentration of oxygen (based on temperature and barometric pressure). Oxygen consumption was measured over a period of 12 min at each swimming speed. Swim tests were performed in clean, dechlorinated tap water at 15°C. The critical swimming speed (UCrit) was determined for each fish using the equation given by Brett (1964): 𝑇

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𝑈𝐶𝑟𝑖𝑡 = 𝑉𝑓 + [( 𝑡 ) 𝑑𝑉] (1),

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where UCrit is in cms-1, Vf is the velocity prior to the velocity at which exhaustion occurred, dV is

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the velocity increment, t is the time swum at each velocity, and T is the time swum at the final

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velocity before exhaustion. N=10 for control, low and high groups. Urine Collection

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Fish were given 24 hours recovery in exposure tanks post swim performance test and

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prior to bladder cannulation to determine renal function. A subset of fish (N= 8 for control, low, and high) were cannulated without prior swim performance test. The cannulation protocol followed Wood and Randall (1973), where fish were anesthetized in a solution of neutral

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buffered 0.1 gL-1 MS-222 (Sigma-Aldrich).

Fish were fitted with internal urinary bladder

catheters (Clay-Adams PE10 tubing, with PE60 sleeves attached by VetBond veterinary glue)

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and the catheter sleeve was stitched onto the base of the tail. Fish were revived by flushing freshwater across the gills and were allowed to recover for 24 hours. Catheters emptied into pre-

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weighed vials and urine samples were collected over 24 hours to measure sodium, potassium, calcium, magnesium, chloride, urea, ammonia, glucose, and protein concentration, and urine flow rate (UFR; N=15). UFR was the total volume of urine collected over 24 h. Glomerular filtration rate (GFR) was determined in a subset of animals (N=8 control and low; N=6 high) by injection of 1 µCi [3H]polyethylene-4000 ([3H]PEG-4000) (Sigma-Alridch) in 1 mL of Cortland saline into the caudal vein at the time of cannulation. After ensuring catheter patency, urine

samples were collected continuously over a 2 hour period.

After cannulation and urine

collection, fish were euthanized and liver, gill, and kidney tissues dissected (described above). Urine and Plasma Analyses Urine and plasma cation concentrations were measured through atomic absorption

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spectroscopy (Varian AA-1275), which utilized 13.5 L air min-1 and 2 L acetylene min-1. To eliminate Na+ interference, 0.2% LaCl3 was used in the Ca2+ and Mg2+ measurements. Urinary ammonia was measured via spectrophotometry using a modified protocol based on phenolhypochlorite method (Verdouw et al. 1978) where indophenol blue is produced by the reaction of ammonia with salicylate and hypochlorite, in the presence of sodium nitroprusside. Urinary urea was measured using the diacetyl monoxime method (Sigma-Aldrich) and chloride concentrations

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for both urine and plasma were measured using the mercuric thiocyanate method (Sigma-

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Aldrich). Glucose concentrations for both urine and plasma were measured using the InfinityTM Glucose Kit (Thermo-Scientific, Wilmington, DE) and urinary protein concentrations were

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measured using Bradford Reagent (Sigma-Alridch) and BSA standards.

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For fish injected with [3H]PEG-4000, 5 mL of Perkin-Elmer Opti-phase scintillation fluid (Waltham, MA, USA) was added to urine samples. [3H]PEG-4000 radioactivity (measured in β

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emissions; counts min-1 ml-1) was determined by using a Tri-Carb 2900TR Liquid Scintillation

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Analyzer (Perkin-Elmer, Waltham, MA, USA). GFR was calculated using the equation given by

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Robertson and Wood (2014):

GFR=

urine (cpm/ml)

initial-final plasma (cpm/ml)

1

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× M × ∆t (2),

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where M is the mass of the fish (g), t is time in hours, and the urine (cpm/ml) and initial-final plasma (cpm/ml) ([initial +final] × 0.5) were based upon measured sample radioactivity.

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Gill Morphology Gill morphological measurements were carried out following protocol outlined by

Hughes (1984). Total number of filaments on each gill arch was counted under a Zeiss Axiolab microscope (Carl Zeiss, Hallbergmoos, Germany), and the length of every tenth filament was measured at 10x magnification using digital image capture and AxioVision Microscope Software (Carl Zeiss, Hallbergmoos, Germany). Linear spacing between lamellae was measured over 10

lamellae at 20x magnification at the base, mid-section, and tip of every tenth filament on the first gill arch. At every tenth filament from the first gill arch, serial cross sections were made with a razor blade and lamellar area was assessed on at least five lamellae from each filament at 20x magnification. Total gill surface area was calculated as A = LnB, where L is the total filament length (mm) on all gill arches, n is the number of lamellae per mm on both sides of the filament,

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and B is the average bilateral surface area of the lamellae (mm2). Enzyme Activity

Flash frozen gill and kidney samples were ground in a mortar and pestle over liquid nitrogen and Na+-K+-ATPase activity was measured following the micro-plate protocol (McCormick, 1993). H+-ATPase activity was measured following a modified protocol from Lin

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and Randall (1993), which was adapted to the Na+-K+-ATPase activity assay (McCormick, 1993)

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using sodium azide and NEM (N-ethylmaleimide) as inhibitors. The plate was monitored in a temperature controlled plate reader at 340 nm for both activities at 15 second intervals over 30

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minutes. Activities were measured as the difference in ATP hydrolysis in the absence and

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presence of an inhibitor (ouabain and sodium azide with NEM), expressed as micromoles of ADP per milligram of protein per hour. Protein concentrations were measured using Bradford

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Reagent (Sigma-Alridch) and BSA standards.

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Liver Glycogen

Frozen liver samples were homogenized in ultrapure water and boiled for five minutes to

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inactivate enzymes. Samples were centrifuged at 13,000xg for 5 minutes to remove insoluble material. Liver glycogen was assessed using a commercially available glycogen assay kit

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(Sigma-Aldrich), following the manufacturer’s protocol.

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Acetaminophen Analysis Acetaminophen was extracted from the water samples in the same manner as Galus et al.

(2013b). Samples were analyzed at the Water Quality Centre at Trent University (ON, Canada) using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESIMS/MS) using an AB Sciex Qtrap 5500 mass spectrometer coupled with a Shimadzu 10A liquid chromatograph instrument and Perkin Elmer Series 200 Autosampler. Compounds were separated using a Thermo Acclaim RSLC120 C18 column (2.2 m, 4.6 x 150 mm). The mobile

phases were methanol and 20 M ammonium acetate in milli-Q water, operated with gradient elution and a flow rate of 0.25 mL/min. Injection volume was 10 L. Acetaminophen was analyzed in positive ion mode and in multiple reaction monitoring mode (MRM), with two precursor-product ion transitions for each analyte. Samples were quantified with internal calibration of the isotopically labelled standard, using a seven- point calibration curve. The

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method limit of quantitation (LOQ) was 0.1 µgL-1. Statistical Analysis

Data was analyzed using SigmaPlot software (version 11, Systat Software, Inc, San Jose, California, USA). Histological data was analyzed based on percent incidence of histological change within each exposure and analyzed using student’s t test. Urine data was analyzed

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together after determining there were no differences between fish that were and were not

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subjected to swim performance tests prior to cannulation. Urine and plasma parameters, liver glycogen, enzyme activity, metabolic rate, and critical swimming speed were analyzed by a one-

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way analysis of variance (ANOVA), comparing all treatments. Homogeneity of variance and

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normality were first assessed by SigmaPlot and the Shapiro-Wilk’s test. Urine potassium, magnesium, calcium, chloride, glucose, protein, plasma calcium, liver glycogen were not normal

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and analyzed using Kruskal-Wallis one-way analysis of variance on ranks. Following ANOVA

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analysis, significant differences between treatments were determined using Tukey’s honest

RESULTS

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Exposure

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significance test (p<0.05). All data is expressed as mean±S.E.M.

Measured acetaminophen concentrations in water were consistent over 72 h and averaged 5.34 ±

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0.08 µgL-1 and 16.4 ± 0.13 µgL-1 in the low (10 µgL-1 nominal) and high (30 µgL-1 nominal) treatments, respectively (see Table S1 for full time course data). Acetaminophen concentrations in control water was below the LOQ (< 0.1 µgL-1). Time Course Histology

Exposure of rainbow trout to acetaminophen at 10 µgL-1 for 2-6 weeks resulted in a significant increase in the incidence of histological changes in the kidney (Table 1). Histological changes observed include movement and loss of nuclei (Fig. S1C,D), non-uniform nuclei size (Fig. S1C,D), non-uniform cytoplasmic staining (Fig. S1B,D), and loss of tubule integrity (Fig. S1B). All exposed fish showed some level of kidney histological change at all sampled time

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points and the severity increased with increased exposure time length (Table 1).

Exposure to 10 µg L-1 acetaminophen for 2-6 weeks also caused a significant increase in incidence of histological changes in the gill as early as two weeks after exposure (Table 2). Histological changes observed included filament end swelling, whole filament swelling, and swelling of the lamellae (Fig. S2B). Exposed fish showed some level of gill histological change

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at all sampled time points (Table 2).

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Exposure of rainbow trout to 10 µgL-1 acetaminophen caused histological changes in the liver as early as 2 weeks. Changes in the exposed group included decreased perisinusoidal space

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Acetaminophen Impacts on Kidney

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and cytoplasm vacuolation.

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Exposure of rainbow trout to acetaminophen at 10 µgL-1 and 30 µgL-1 for four weeks resulted in similar histological changes as seen in the kidney after the time course exposure (Fig.

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1). Interestingly, the histological changes, such as non-uniform cytoplasmic staining and loss of tubule integrity, seen in the high dose group were similar to changes found after 6-week

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exposure to 10 µgL-1 acetaminophen (Fig. S1). Movement and loss of nuclei and non-uniform nuclei size were more common in the 10 µgL-1 group, while the 30 µgL-1 group showed non-

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uniform cytoplasmic staining and loss of tubule integrity in addition to the changes seen in the low dose group. Thus, the severity of histological change increased with increasing exposure concentration. Urine filtration rate (UFR) increased with increasing acetaminophen exposure

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concentration, although there was no difference was seen in glomerular filtration rate (GFR) across treatments (Fig. 2). Sodium, chloride, potassium, and calcium concentrations were increased in the urine of both exposure groups and differences were greater in the higher exposure concentration (Fig. 3A). Magnesium concentration did not change with acetaminophen exposure (Fig. 3A). Urea and ammonia concentrations increased in the urine of both exposure groups (Fig. 3B). Likewise, urinary protein and glucose concentrations were elevated with

exposure although only glucose concentrations were different between low and high doses (Fig. 3C). Kidney Na+-K+-ATPase (NKA, Fig. 4) and H+-ATPase (Fig. S3) activity was not different across treatment groups. Acetaminophen Impacts on Gill

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Exposure of rainbow trout to acetaminophen at 10 µgL-1 and 30 µgL-1 for 4 weeks resulted in similar histological changes seen in the gill (Fig. 5) as in the time course exposure (Fig. S2). The high dose group showed changes in the gill that were similar to changes seen with 6-week exposure to 10 µgL-1 in the time course exposure. Lamellar swelling was found in both exposure groups, however, filament tip swelling was found at 10 µgL-1 and whole filament swelling was found at 30 µgL-1. Gill morphometric analysis revealed that lamellar spacing decreased in both

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low and high exposure groups and lamellar area was decreased in the high exposure group but

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not in the low exposure group (Table 3). There were no significant changes for total gill surface

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with exposure (Table 3).

Oxygen consumption was reduced in trout from the acetaminophen treatments at the two

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fastest swimming speeds (2.5 and 3.0 BL/s). At both these speeds, the impact was greater in fish from the higher acetaminophen exposure treatment (Fig. 6). Maximum oxygen consumption for

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the control, low and high group were 640.8, 514.9, and 453.8 mg O2 kg-1 hr-1, respectively.

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Critical swimming speed (Ucrit) was significantly lower in the exposure groups compared to unexposed fish (Fig. 7). NKA activity was elevated only in the high dose group (Fig. 4) but H+-

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ATPase activity was not significantly different with acetaminophen exposure compared to controls (Fig. S3).

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Acetaminophen Impacts on the Liver Exposure of rainbow trout to acetaminophen to 10 µgL-1 and 30 µgL-1 resulted in

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increased histological changes in the liver (Fig. 8). Decreases in the perisinusoidal space were seen in both the low (Fig. 8B) and high (Fig. 8C) acetaminophen concentrations. Cytoplasmic vacuolation was observed in the high exposure group only (Fig. 8C). Liver glycogen content decreased in the high exposure group (Fig. 9). Plasma Ion and Glucose Concentrations

Plasma concentrations of sodium, chloride, potassium, calcium, or magnesium were similar across exposure groups (Fig. S4A). Similarly, plasma glucose concentrations did not change across exposure groups (Fig. S4B). DISCUSSION

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While histological effects of a variety of analgesics have been previously observed in three fish species, the functional implications of these changes had not been determined. Presently, we have observed both histological and functional changes in the kidney, gill and liver of rainbow trout with acetaminophen exposure. Acetaminophen Impacts on Kidney

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Exposure to 10 µgL-1 acetaminophen caused histological changes in the kidney as early as 2 weeks, and the severity of the changes increased with increasing exposure time. The

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histological effects were consistent with other studies on analgesics including acetaminophen

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(Schwaiger et al., 2004; Triebskorn et al., 2004; Galus et al. 2013b). For instance, rainbow trout

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exposed to 5 µgL-1 diclofenac showed changes in tubular epithelial cells and non-uniform cytoplasmic staining comparable to the changes seen in this study (Schwaiger et al., 2004).

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Zebrafish exposed to 0.5 and 10 µgL-1 acetaminophen showed comparable changes, including structural alterations in to the proximal kidney tubule structure, alterations in nuclear size or

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density, plasma alterations, loss of staining integrity and the presence of hyaline or hypertrophic/hyperplasic tissue (Galus et al., 2013b). However, no changes in distal kidney

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tubule structure were seen in zebrafish (Galus et al., 2013b). In the teleost kidney, urine formation involves glomerular ultrafiltration and substantial

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reabsorption of filtered substances across the tubular epithelial (Marshall & Grosell, 2005). The majority of monovalent ions are reabsorbed in the water-impermeable distal tubules. In the early

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distal tubules, sodium and chloride in the lumen creates a lumen positive potential that is maintained by the basolateral NKA and apical Na+-K+-Cl--cotransporter (NKCC), which allows for uptake of ions including sodium, chloride, and potassium (Dantzler, 2003; Nishimura et al., 1983). Glucose and other organic solutes are reabsorbed in the proximal tubules, while larger substances, such as proteins, are generally not filtered and remain in the plasma (Braun & Dantzler, 1997). In order to understand how the histological changes observed in rainbow trout

exposed to acetaminophen might impact kidney function, urine composition and glomerular filtration rate (GFR) were determined. Acetaminophen increased urine filtration rate (UFR) and urinary concentrations of sodium, potassium, calcium, chloride, ammonia, urea, protein, and glucose suggesting that the kidneys from exposed fish may have reduced capacity to reabsorb filtered substances in both the proximal and distal tubules and a loss of filtration selectivity.

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Acetaminophen overdose in human patients has been shown to cause renal insufficiency characterized by tubular epithelial cell necrosis in both the proximal and distal tubules (Bjorck et al., 1988). The fact that GFR did not change with exposure (Fig. 4) suggests that reabsorption was being impacted rather than increased kidney filtration causing a rise in urinary ion and glucose concentrations. However, the presence of protein in the urine may also indicate glomerular damage. Pharmaceutical effects on the glomerulus of fish has not been extensively

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studied, however in a diclofenac exposure study on rainbow trout, the authors argued that the

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accumulation of hyaline protein droplets could be a result of impaired glomerular filtration (Triebskorn et al., 2004). Similar histological changes in the kidney have been seen in aquatic

(Benli et al., 2008).

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Acetaminophen Impacts on Gill

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species as a result of metal (Mela et al., 2007; Gupta & Srivastava, 2006) or ammonia exposure

Exposure of rainbow trout to 10 µgL-1 acetaminophen caused histological changes in the

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gill as early as 2 weeks and the incidence of histological effects was greater with exposure compared to control; the severity of the histological changes increased with increasing exposure

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time. Similar changes were seen in the gills of rainbow trout exposed to 5 µgL-1 of diclofenac including inflammation of the lamellae and filament, however, we did not observe the epithelial

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cell necrosis and pillar cell death that has been seen in diclofenac exposure studies (Schwaiger et al., 2004). Interestingly, no changes in the gill were observed in zebrafish exposed to

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acetaminophen (Galus et al., 2013b), suggesting that the zebrafish gill may be less sensitive to analgesic induced histological effects. Oxygen consumption increased exponentially with increasing swimming speed in control fish, similar to previous studies with rainbow trout (Alsop and Wood, 1997). However, rainbow trout exposed to acetaminophen showed a reduced capacity to take up oxygen from the water at higher swimming speeds. Additionally, critical swimming speed was reduced in both exposure

groups, indicating that the histological changes seen in the gills may be limiting oxygen uptake. Gill morphometric analysis showed that lamellar density decreased in both exposure groups and lamellar area was decreased in only the high group (Table 3). Reduced swimming performance and structural changes in the gills after exposure to a variety of contaminants have been reported in the past (Boyle et al., 2013; Ellgaard & Guillot, 1988; Nikl & Farrell, 1993; Wilson & Wood,

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1992; Waiwood & Beamish, 1978). To our knowledge, this has not yet been reported after pharmaceutical exposure.

Although not measured, the swelling of the filaments and lamellae seen in the histology could increase the diffusion distance oxygen needs to travel between the blood and the environment. Gill morphometric analyses indicated decreased lamellar spacing and lamellar area,

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which would decrease oxygen uptake. Both of these changes may explain the lowered swimming performance with acetaminophen exposure. However, morphological changes seen may be a

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result of gill remodeling to minimize ion loss. For instance, freshwater fish have been known to

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undergo gill remodeling when faced with salinity or hypoxic challenges in order to maintain ion

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homeostasis (Nilsson, 2007; Mitrovic et al., 2009; Goss et al., 1994). Whether or not the decreased oxygen consumption is a result of the histological changes due to acetaminophen exposure or an indirect effect due to gill remodeling in order to minimize ion loss at the gills is

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not clear.

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Acetaminophen Impacts on Liver

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Exposure of rainbow trout to 10 µgL-1 acetaminophen caused histological changes in the liver as early as 2 weeks. Although the changes were not scored for severity, changes seen in the exposed group included decreases in perisinusoidal space and cytoplasm vacuolation, with the

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latter only present in the high exposure group. In zebrafish exposed to acetaminophen, the liver showed cytoplasmic vacuolization that was thought to indicate a decrease in glycogen stores

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(Galus et al., 2013b). However, increased size of hepatic nuclei was also observed in exposed zebrafish although they were not in the present study with rainbow trout. Histological changes were also seen in the liver of diclofenac exposed rainbow trout (Schwaiger et al., 2004). Whether the variation in liver histology effects is due to a difference in species (rainbow trout versus zebrafish), compound (diclofenac versus acetaminophen), or both remains to be determined.

Further research on liver effects of analgesics will be required to determine the hepatic effects of compounds in this drug class. Histological changes in the liver following pharmaceutical exposure are not surprising as it is the primary xenobiotic metabolizing organ and liver damage is observed in human patients

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who have experienced acetaminophen overdose (Mazer and Perrone, 2008). The vacuolation in the high dose group could be caused by glycogen depletion and/or provide evidence for metabolic disturbances (Woodward et al., 1994; Figueiredo-Silva et al., 2005). Indeed, liver glycogen stores were lower in fish from the highest acetaminophen exposure group only, supporting this interpretation of the histology. These changes are consistent with a general type of cellular response to stress factors and the collapse of cellular compartmentalization has been

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shown in other xenobiotic exposure studies in fish (Schramm et al., 1998, Gernhöfer et al., 2001,

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Triebskorn et al., 2004).

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Maintaining Homeostasis with Altered Organ Function

Our results showed that after acetaminophen exposure, rainbow trout experience

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increased loss of ions and glucose in urine, which could impact plasma concentrations. However, since no exposure-related mortalities were seen, we hypothesized that the fish were able to

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maintain homeostasis. Indeed, plasma concentrations of sodium, chloride, potassium, calcium,

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magnesium, and glucose in acetaminophen exposed fish were not different from controls. A variety of studies have shown the uptake of sodium and chloride are via transporters such as Na+-

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K+-ATPase (NKA) and H+-ATPase (Lin et al., 2004; Richards et al., 2003; Reid et al., 2002; Marshall & Grosell, 2005). In the kidney, no changes in NKA or H+-ATPase activity were seen in either of the exposure groups (Fig. 4, Fig. S3), possibly indicating a lack of ability to maintain

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reabsorption homeostasis in the kidney. In the gill, enzyme activity data supports the notion of some compensation at the gill as NKA activity was increased in the gill at the high dose,

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although H+-ATPase activity did not change (Fig. 4, Fig. S3). It is possible that the fish could be compensating for the loss of ions through other ion-absorbing organs, such as the intestines (D’Cruz & Wood, 1998; Wood et al., 2002; Marshall & Grosell, 2005) and bladder (Marshall & Grosell, 2005; McDonald et al., 2002). Like plasma ion concentrations, plasma glucose was not altered with acetaminophen exposure. To explore compensation for glucose loss, liver glycogen content was assessed.

While no change was found in the low exposure group, a significant decrease in liver glycogen was found in the high dose group (Fig. 9), suggesting that liver glycogen breakdown may partially contribute to maintaining plasma glucose levels Implications for Fish Health

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We have documented that the histological changes caused by acetaminophen coincide with changes in kidney and gill function. Altered gill histology and morphology were found in fish with acetaminophen exposure; these fish had lowered oxygen consumption and critical swimming speed compared to the controls. Although it is unclear if acetaminophen is directly impacting aerobic metabolism, there are disadvantages to relying on anaerobic metabolism, such as lactate build-up and metabolic acidosis. Limitations to oxygen consumption and swimming

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performance could have an impact on growth, predator-prey interactions, and behaviour (Van

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Raaij et al., 1996; Pichavant et al., 2001).

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Altered kidney histology and changes in urine composition, coupled with a lack of impact on glomerular filtration rates, strongly suggests that acetaminophen induced damage to the

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proximal and distal kidney tubules were impacting the capacity of the fish to reabsorb ions and glucose. In spite of these functional changes, fish were able to maintain plasma ion and glucose

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concentrations, however, this most likely occurs at the cost of increased energy expenditure. This

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could lead to perturbations in ion and nutrient homeostasis that may have long-term impacts on growth, metabolism, reproduction, and a host of other cellular processes (Morgan & Iwama,

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1991; Swanson, 1998; Lambert et al., 1994; Febry & Lutz, 1987), and impact stress responses (Pottinger & Carrick, 1999) and feeding behaviors (Cowey et al., 1977; Panserat et al., 2000;

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Moon, 2001).

Analgesics are one of the most common environmental pharmaceutical classes, and data

from this study and others suggests that analgesics in the environment, such as acetaminophen,

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may impact gill, liver and kidney histology; data from this study suggest that organ function is also compromised. More research is needed to understand if these impacts are present in fish exposed to pharmaceuticals in the wild and if the effects are significant enough to impact long term processes of growth, metabolism, and reproduction.

Acknowledgements The authors would like to thank members of the Wilson lab for helping with fish care and experiment set-up. We would also like to thank Alex Zimmer and Tamzin Blewett for helping with enzyme activity assays, Kyle Crans and Brittney Borowiec for helping with the swim tunnel

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experiments, and Lisa Robertson for helping with bladder cannulations. The [3H]PEG-4000 was generously donated by Dr. Chris Wood (McMaster University). This research was funded by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Program and the Ontario Ministry of Research and Innovation’s Early Research

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Award Program to J.Y.W.

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Boyle, D., Al-Bairuty, G.A., Ramsden, C.S., Sloman, K.A., Henry, T.B., Handy, R.D., 2013. Subtle alterations in swimming speed distributions of rainbow trout exposed to titanium dioxide nanoparticles are associated with gill rather than brain injury. Aquat. Toxicol. 126, 116-27.

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TABLES

1 4 4 7 0 0 0

2 2 0 2 6 8 4

3 0 0 0 6 0 2

4 Incidence 0 0.6 0 0.5 0 0.75 0 1 0 1 6 1

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0 4 4 3 0 0 0

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CTRL Week 2 CTRL Week 4 CTRL Week 6 ACE Week 2 ACE Week 4 ACE Week 6

N 6 6 6 6 6 6

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Table 1. Rainbow trout were exposed to 10 µgL-1 acetaminophen (ACE) for 2, 4 or 6 weeks. Histological scores were adapted from Bernet et al. (1999). Kidney histological changes were scored from 0-4, 0 representing no damage to the kidney, 1 representing nuclei movement, 2 for increased nuclei movement and multi-nucleation and loss of nuclei, 3 for heavy globular eosin staining and tubule damage, and 4 for heavy tubule degradation. All ACE exposure times showed a statistically significant increase in incident of histological change, when compared to controls; p≤0.05.

N 6 6 6 6 6 6

0 3 3 1 0 0 0

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CTRL Week 2 CTRL Week 4 CTRL Week 6 ACE Week 2 ACE Week 4 ACE Week 6

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Table 2. Rainbow trout were exposed to 10 µgL-1 acetaminophen (ACE) for 2, 4 or 6 weeks. Gill histological changes were scored on a scoring system from 0-3 based on number of histological changes (which include filament tip swelling, thickening of lamellae, and whole filament swelling) observed; 0 indicating no histological., 1 indicating at least one of the changes were observed, 2 indicating at least two of the changes were observed, and 3 indicating all three possible histological changes were observed. All exposure times showed a statistically significant increase in incident of histological changes, when compared to controls; p≤0.05. 1 3 3 5 0 1 0

2 0 0 0 4 3 4

3 Incidence 0 0.6 0 0.5 0 0.83 2 1 2 1 2 1

Low 366.1±5.9 2.14±0.06 128.1±2.3

53.67±0.82 A

48.54±1.01B

0.141±0.004

0.140±0.001A

0.130±0.002B

104.5±3.2

101.7±5.3

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Control 365.5±6.9 2.07±0.12 131.0±5.0

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Filament number Filament length (mm) Filament thickness (µm) Lamellar density (lamellae/mm filament) Lamellar area (mm2)

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116.3±11.3

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Total surface area (cm2)

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Table 3. Gill morphometric s of rainbow trout exposed to 0 µgL-1 (control), 10 µgL-1 (low), and 30 µgL-1 (high) acetaminophen for 4 weeks. Linear spacing between lamellae was measured over 10 lamellae at 20x magnification at the base, mid-section, and tip of every tenth filament on the first gill arch. At every tenth filament from the first gill arch, serial cross sections were made with a razor blade and lamellar area was assessed on at least five lamellae from each filament at 20x magnification. Total gill surface area was calculated as A = LnB, where L is the total filament length (mm) on all gill arches, n is the number of lamellae per mm on both sides of the filament, and B is the average bilateral surface area of the lamellae (mm2). Differences between treatments were determined with a one-way ANOVA followed by Tukey’s honest significance test to determine differences among groups (p<0.05). Different letters represent significant differences between groups (N=8 for control and high; N=7 for low). High 372.5±5.9 2.09±0.06 132.8±6.0

50.65±0.27B

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Figure 1. Histological alterations in the kidney after a 4 week exposure to 0, 10, or 30 µgL-1 acetaminophen. All slides were screened at 60x under a standard light microscope. A) Normal kidney tubules of rainbow trout showing central nuclei positioning with a uniformly stained cytoplasm and uniform nuclei size. B) Kidney tubules of rainbow trout exposed to 10 µgL-1 of acetaminophen, showing loss of nuclei (proximal tubule, open white arrow), nuclei movement (proximal tubule, open black arrow), and multi-nucleation (distal tubule, black arrow). C) Proximal kidney tubules of rainbow trout exposed to 30 µgL-1 acetaminophen, showing nonuniform cytoplasmic staining (proximal tubule, black chevron).

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Figure 2. Urine Filtration Rate (UFR) and Glomerular Filtration Rate (GFR) in rainbow trout exposed to 0 µgL-1 (control), 10 µgL-1 (low), and 30 µgL-1 (high) acetaminophen for 4 weeks. Significant differences between groups was determined using a one-way ANOVA followed by Tukey’s honest significance test to determine differences among groups (p<0.05). Error bars depict standard error of mean (N=8 for control and low; N=6 for high). Different letters over bars indicate statistical differences amongst treatment groups.

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Figure 3. A) Cation (Na+, K+, Mg2+, Ca2+), anion (Cl-), B) ammonia, urea, C) glucose, and protein concentrations in urine of rainbow trout exposed to 0 µgL-1 (control), 10 µgL-1 (low), and 30 µgL-1 (high) acetaminophen for 4 weeks. Significant changes were determined using a oneway ANOVA followed by Tukey’s honest significance test to identify specific differences between treatments (p<0.05). Error bars depict standard error of mean. (N=15 for each group). Different letters over bars indicate statistical differences amongst treatment groups within a measured parameter.

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Figure 4. Na+-K+-ATPase (NKA) activity in kidney and gill of rainbow trout exposed to 0 µgL-1 (control), 10 µgL-1 (low), and 30 µgL-1 (high) acetaminophen for 4 weeks. Significance between groups was determined using a one-way ANOVA followed by Tukey’s honest significance test to determine differences among groups (p<0.05). Asterisk represents significant difference from control. Error bars depict standard error of mean (N=10 per group).

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Figure 5. Histological alterations in the gills of rainbow trout after 4 week exposure to acetaminophen. All slides were screened at 60x under a standard light microscope. A) Normal gill filament of rainbow trout showing uniform thin filaments with normal lamellae. B) Gill filament of rainbow trout exposed to 10 µgL-1 of acetaminophen, showing both enlargement of the filament tip (black arrow), along with swelling of the lamellae (open black arrow). C) Gill filament of rainbow trout exposed to 30 µgL-1 of acetaminophen, showing enlargement of the lamellae (open black arrow).

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Figure 6. Oxygen consumption versus swimming speed (body lengths per second; BL/s) of rainbow trout exposed to 0 µgL-1 (control), 10 µgL-1 (low), and 30 µgL-1 (high) acetaminophen for 4 weeks. Oxygen consumption data points at a given swimming speed with different letters are significantly different as determined by ANOVA followed by Tukey’s honest significance test to determine differences among groups (p<0.05). Error bars depict standard error of mean (N=8 per group).

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and 30 µgL-1 (high) acetaminophen for 4 weeks. Significant differences between groups were determined using a one-way ANOVA followed by Tukey’s honest significance test to determine differences among groups (p<0.05). Asterisk represents significant difference from control. Error bars depict standard error of mean (N=10 per group).

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Figure 8. Histological alterations were observed in the liver after 4 week exposure to 0, 10, or 30 µgL-1 acetaminophen. All slides were screened at 60x under a standard light microscope. A) Normal liver of rainbow trout showing normal perisinusoidal space. B) Liver of rainbow trout exposed to 10 µgL-1 of acetaminophen, showing decreased perisinusoidal space (black arrow). C) Liver of rainbow trout exposed to 30 µgL-1 of acetaminophen, showing cytoplasmic vacuolation (open arrow).

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Figure 9. Liver glycogen content of rainbow trout exposed to 0 µgL-1 (control), 10 µgL-1 (low), and 30 µgL-1 (high) acetaminophen for 4 weeks. Significant differences between groups was determined with a one-way ANOVA followed by Tukey’s honest significance test to determine differences among groups (p<0.05). Asterisk represents significant difference from control. Error bars depict standard error of mean (N=10 per group).